1. Field of the Invention
The present invention relates to the formation of crystals of small and large molecules, including macromolecules such as proteins and nucleic acids, and more particularly, to a method and apparatus for dynamically controlling the process of forming such crystals, and the resulting crystals.
2. Description of the Related Art
There is great interest in determining the three-dimensional (3-D) structures of biological molecules. Ongoing studies of the genomes of humans and other mammals, as well as of disease-causing viruses, bacteria, and parasites, are identifying thousands of genes and proteins, many of which are linked to diseases of humans and domestic animals and plants. Knowing the 3-D structures of the molecules that are involved in causing a disease can be a tremendous aid in developing drugs to prevent or treat the disease. Rational drug design involves obtaining a precise 3-D structure of a specific molecule involved in a disease process, making and studying physical models and computer-generated graphic images of the structure, as well as using sophisticated computer programs that thermodynamically model the structure and its interactions with solvent and other molecules, in order to design drugs that selectively bind to and alter the function of the disease-causing molecule. Rational drug design can be used to analyze and design drugs that interact with small molecules such as peptide and non-peptide hormones, as well as intermediate-sized and large macromolecules such as nucleic acids and proteins, respectively. The application of rational drug design will result in a number of diseases and pathological symptoms being brought under control. For example, CAPTOPRIL is a well known drug for controlling hypertension that was developed through rational drug design techniques. CAPTOPRIL inhibits generation of the angiotension-converting enzyme thereby preventing the constriction of blood vessels.
X-ray crystallography is a technology that allows us to obtain the precise 3-D atomic structures of molecules such as peptides, proteins, and nucleic acids. A critical step in using X-ray crystallography to determine the 3-D structure of any molecule of interest is establishing a reliable method for crystallizing the molecule. Proteins are one of the major classes of structural molecules in living organisms; protein enzymes catalyze the metabolic reactions that make life possible; and many disease processes are mediated by the interactions of proteins with other molecules, including other proteins. Therefore, much time and money have been spent crystallizing proteins for analysis of their structures. Recent biotechnological developments in cloning and over-expression of genes encoding proteins of interest and in the purification of proteins are increasing the need for a reliable way in which to grow protein crystals. Unfortunately protein crystallization is a difficult and unpredictable art.
Crystallization of a biological molecule such as a protein involves the creation of a supersaturated solution of the molecule under conditions that promote minimum solubility and the orderly transition of the molecules from the solution into a crystal lattice. The variables that must be controlled precisely to promote crystal growth include temperature, protein solution concentration, salt solution concentration, pH, and gravitational field, for example (Durbin, S. and Feher, G. Ann Rev Phys Chem 47 (1996) 171-204, the entire contents of which are incorporated herein by reference). These variables are carefully controlled and optimum combinations thereof are determined through experimentation to yield superior crystals.
In the description of the invention that follows, the molecule being crystallized, e.g., the protein, is referred to as a xe2x80x9creactant,xe2x80x9d and the solution containing one or more precipitants that is used in crystallization processes is referred to as a xe2x80x9creagentxe2x80x9d solution.
The crystallization process generally involves three distinct phases; nucleation, sustained crystal growth, and termination of crystal growth. Nucleation is the initial formation of an ordered grouping of a few reactant molecules and requires a particular concentration of reactant molecules in a precipitating reagent solution. On the other hand, the continued growth phase consists of the addition of reactant molecules to the growing faces of the crystal lattice and requires lower concentrations of reagent solution than the nucleation phase. The termination phase can be initiated by poisoning the growing lattice with denatured or chemically modified reactant molecules or with different molecules, by depletion of the reactant solution, or by changing the concentration of precipitant to a specified level.
It is considered desirable to obtain a small number of crystallization nuclei quickly that will grow slowly into full-sized crystals. Theoretically, this allows for a relatively large size of the resulting crystals, homogenous crystal order and morphology, and balanced crystal dimensions. Therefore, it is desirable to begin crystallization with a reagent solution containing a particular concentration of precipitant until nucleation is detected, at which point it is desirable to adjust the concentration of precipitant. Thus, one of the critical requirements of any molecular crystallization process is the fine and dynamic control of the various parameters that determine the concentration of the precipitant in the solution in which the target reactant molecule protein is suspended. This control requires the ability to attain nucleation conditions and the ability to modify the concentration of the precipitant without disturbing the crystallization process.
There are several conventional techniques for forming molecular crystals; for example, liquid diffusion, vapor diffusion and dialysis techniques. These processes are relatively slow and cannot be readily controlled dynamically. Therefore, these processes require complex and large apparatus in order to control crystallization, if crystallization can be controlled at all. Accordingly, it is desirable to overcome these limitations.
Most conventional protein crystallization methods mix a solution containing the reactant protein molecules with a crystallizing, or precipitating, solution to accomplish crystallization. In terms of mechanics, use is commonly made of syringes, stepping motors, valves of various types, membranes to separate solutions, and in one case, a gel to replace the membrane and act as a delaying filter device between solutions. U.S. Pat. Nos. 4,917,707, 5,106,592, and 5,641,681, and Microdialysis Crystallization Chamber, L. C. Sieker, J. Crystal Growth 90 (1988) 349-357, the entire contents of which are incorporated herein by reference, disclose these concepts.
It is known to xe2x80x9ccontrolxe2x80x9d the crystallization process. However, only the movement of liquids via pumps, valves and syringes is controlled in conventional crystallization processes. This control creates a static condition (bath concentration) which is predefined for the protein in question. For example, U.S. Pat. No. 4,755,363, the entire contents of which are incorporated herein by reference, discloses delivering liquids at desired flow rates and concentrations. However, U.S. Pat. No. 4,755,363 fails to disclose changing conditions within the crystallization chamber (with the exception of temperature) once those conditions have been set and crystallization has begun.
Temperature is an important parameter that can be controlled to optimize conditions separately for nucleation or growth. U.S. Pat. Nos. 4,755,363 and 5,362,325, the entire contents of which are incorporated herein by reference, are exemplary of patents disclosing temperature control in crystallization processes. U.S. Pat. No. 5,362,325 discloses varying the concentration of a crystallizing agent over time to produce a predetermined gradient in the concentration of the crystallizing agent. However, this reference fails to disclose dynamic control as disclosed for this invention.
Automation is a recent trend in crystallography. Robotics enables systematic pipetting of solutions and protein into crystal growth chambers on plates, so that a multiplicity of conditions can be examined more quickly and consistently. The use of robotics frees valuable time for researchers. Another trend is the use of semi-automated techniques to record results.
Also, inherent to any crystallization process that proceeds in a finite gravitational field are the effects of molecular convection, thermal effects, sedimentation, and buoyancy. Crystallization experiments that have been conducted in microgravity (1/1000 g to 1/10,000 g) on board the Space Shuttle, Space Station, and other vehicles, indicate that larger and more homogenous crystals can be grown in microgravity environments by eliminating the effects of the earth""s gravitational field. Several patents disclose crystallization in microgravity to improve the size, morphology and diffraction quality of crystals. U.S. Pat. Nos. 5,362,325 and 4,755,363, which are incorporated herein by reference, are exemplary of patents disclosing microgravity crystallization. In fact, a low gravity environment is xe2x80x9ccrucialxe2x80x9d (emphasis added) to the success of one apparatus (Sygusch, et al. J. Crystal Growth 162 (1996) 167-172). However, the practical limitations of using current space vehicles, such as the Space Shuttle, render it difficult to use conventional apparatus/methods for crystallization in microgravity environments. Particularly, conventional apparatus are too large, are difficult to control remotely and automatically, have many moving parts that can fail, do not permit accurate change of solution concentration during the process and are not entirely reusable. Also, known processes require too much time to produce fully grown crystals. Therefore, it is desirable to overcome these deficiencies to permit crystal growth experiments in space under conditions in which gravity ranges below earth gravity down to microgravity conditions. Also, conventional apparatus and methods do not facilitate experimentation in which the only variable is the presence or absence of the earth""s gravitational field because conventional crystallization apparatus must be modified significantly for use in space. Given the unpredictable, multifactorial nature of the crystallization process, and its dependence on the structure of the molecule which is being crystallized, it is also envisioned that some molecules will crystallize efficiently in gravitational fields that are greater than earth""s gravitational field. The foregoing methods and apparatus do not provide a dynamic control capability, either in the earth""s gravitational field or in gravitational fields that are greater or lesser than earth""s gravitational field.
The advantages obtained by growing crystals in reduced gravity in space can also be obtained on the earth""s surface by growing crystals under conditions in which the effective gravitational field (geff) is reduced as a result of diamagnetic effects induced by a very high magnetic field. Provided that a magnet of sufficient strength is used, a solution containing reactant molecules can be moved to positions with respect to the center of the magnet so that the reactant molecules experience geff forces that range between zero and twenty times normal earth gravity (E. Beaugnon et al., Nature (1991) 349:470). A study of lysozyme crystallization under conditions where geff was varied magnetically from 0.95 to 1.05 g suggested that crystal growth is enhanced in a strong magnetic field, possibly because alignment of the reactant molecules in the magnetic field leads to formation of more ordered crystals (N. I. Wakayama et al., J. Crystal Growth (1997) 178:653; see also, G. Sazaki et al., J. Crystal Growth (1997) 173:231). None of the foregoing references discloses methods or apparatus that provide a dynamic control capability for crystallization under magnetically-induced effective gravitational fields where geff is greater or lesser than normal earth gravity.
The present invention is a Dynamically Controlled Crystallization System (DCCS) that includes apparatus and methods that can be used to control a molecular crystallization process precisely and dynamically.
The present invention can be further used to monitor and control a molecular crystallization process remotely in real time.
The present invention can be further used to control a molecular crystallization process in a predetermined manner.
The present invention permits initiation, termination or reversal of a molecular crystallization process.
The present invention permits reduction in the size and number of moving parts in a molecular crystallization method and apparatus.
The present invention permits production of molecular crystals in a sealed system.
The present invention can be used to produce molecular crystals under conditions of real and simulated gravity in the range of from about zero times the earth""s gravitational field to about 20 times the earth""s gravitational field, and to produce crystals in earth""s gravitational field, with identical apparatus.
The present invention permits crystallization parameters to be controlled in a sealed system without changing the pressure in the crystallization chamber.
The present invention can be used to optimize the time required to produce large, well-ordered molecular crystals.
The present invention can be used to optimize crystallization conditions while using less experiments and substantially less reactant, e.g. protein.
xe2x80x9cDynamic controlxe2x80x9d as used herein means that: (1) the crystallization process can be started at will, (2) several conditions that are important for the growth of molecular crystals, such as pH, conductivity, and temperature, can be measured in the crystallization chamber, (3) decisions regarding altering the conditions affecting crystal growth can be made based upon monitoring crystal nucleation and growth, (4) these conditions can be precisely controlled at any time during the crystallization process, (5) the crystallization process can be modified or stopped when desired and (6) the process can be reversed if so desired.
In order to achieve these objectives, a first aspect of the invention includes at least one crystallization chamber for holding a solution containing reactant molecules that is separated by a permeable membrane from a reagent solution comprising a precipitant at a first concentration, at least one source reservoir comprising reagent solution having precipitant at a second concentration that is coupled via a communication passage with the reagent solution in the crystallization chamber, and at least one drain reservoir that is coupled via a separate communication passage with the reagent solution in the crystallization chamber, and transfer mechanisms configured to respectively transfer reagent solution between the at least one source reagent solution reservoir and the crystallization chamber, and to concurrently transfer reagent solution from the crystallization chamber to the at least one drain reservoir. The transfer mechanisms can work in tandem to simultaneously transfer equal amounts of precipitating solution into and out of the crystallization chamber, to thereby alter the concentration of precipitant in the reagent solution in the crystallization chamber with respect to time. In this manner, the concentration of the precipitant in the reagent solution in the crystallization chamber can be adjusted dynamically to provide optimum crystallization conditions with respect to time.
The invention can also be configured so that two or more pairs of source/drain reservoirs, each containing a different reagent solution, are respectively coupled via separate solution communication means to the reagent solution in the crystallization chamber, with separate solution transfer means to transfer reagent solutions tandemly from the source reservoir to the crystallization chamber, and from the crystallization chamber to the respective drain reservoir, for each pair of reagent reservoirs. The paired source and drain reservoirs can be operated sequentially, i.e., one pair at a time, with time allowed for diffusion-mediated mixing of the reagent solution in the crystallization chamber between transfers, so that precipitant concentration in the reagent and reactant solutions in the crystallization chamber is changed in an even, controlled manner.
The invention also can be configured with two or more crystallization chambers coupled in series via solution communication means, with at least one source reagent solution reservoir being coupled to the reagent solution of the first crystallization chamber in the series, and at least one drain reservoir being coupled to the reagent solution of the last crystallization chamber in the series. Tandem transfer of a volume of reagent solution from a source reservoir to the first crystallization chamber, and from the last crystallization chamber to a drain reservoir, results in transfer of an equal volume of reagent solution from each crystallization chamber in the series to the next one, thereby establishing a concentration gradient of a precipitant in the reagent solution across the series of crystallization chambers.
The invention can also be configured so that two or more crystallization chambers are coupled via a manifold solution communication means to at least one source reagent solution reservoir, and are also coupled via solution communication means, directly or indirectly, to one or more drain reservoirs, so that the same volume of reagent solution can be transferred simultaneously from the source reservoir to each of the two or more different crystallization chambers.
By configuring crystallization chambers according to the invention so that the reactants in different crystallization chambers experience a range of different precipitant concentrations and other parameters that affect crystallization; e.g., in multiple series, or in 2- and 3-dimensional arrays, the invention permits controlled variation of the concentrations of one or more precipitants during the process, resulting in establishing conditions for crystal growth, optimization, termination or reversal of the crystallization process, and conservation of the molecular crystal. The invention thus permits the crystallization process to be controlled easily, merely by operating the transfer mechanisms.
Another important feature of the present invention is that it has real time remote-controlled operative capabilities, and can easily be used under normal earth gravitational conditions, as well as in environments in which the real or simulated gravitational field is greater or less than earth""s gravitational field.
The invention also permits a complex set of variables in the process of crystal growth to be rigorously examined, manipulated and controlled so that gravity is the only variable. Due to its compactness and ease of operation, the present invention is conservative in terms of cost containment for flights aboard the Space Shuttle and the International Space Station. Use of this invention conserves both money and valuable astronaut time. The present invention can remain on a space platform for many sets of experiments, while the crystallization chamber can return to earth with the molecular crystals that are produced in space. New chambers, tubing and syringes can be delivered on the next flight pre-loaded. Furthermore, the invention can be readily miniaturized so that it is even more compact than the embodiments shown in the examples disclosed herein, and thus the entire unit can be made even less expensive to transport to and from space.
The present invention further provides methods for carrying out dynamically controlled crystallization of proteins and other molecules under conditions in which the effective gravitational force resulting from diamagnetism in a high magnetic field (i.e., 16 Tesla or more) is in the range of zero to 20 times earth""s gravitational field.
Another important feature of the invention is that the apparatus is compact and easily sealed to the air. Sealing the system permits operation without adverse effects on fluid flow and the sieving properties of the dialysis membrane in the crystallization chamber being caused by changes in air pressure. Sealing also prevents contamination of the solutions in the system sample for further experimentation.
An additional feature of the invention is that it produces crystals in a reduced time with few moving parts and low power requirements.